1. Field of the Invention
The present invention generally concerns the preparation of gels, typically agarose gels, that are suitable to receive samples upon which electrophoresis is performed.
The present invention particularly concerns the placement of precision arrays of slits in trays of gels, typically agarose gels, that are suitable to receive samples upon which electrophoresis is performed.
The present invention still more particularly concerns a spacer-comb element--particularly as is useful in a special apparatus and a method for the easy simultaneous preparation of multiple trays of gels, typically agarose gels, that are suitable to receive samples upon which electrophoresis is performed--that is useful in conveniently placing arrayed slits in the gel of a gel tray.
2. Description of the Prior Art
Agarose gel electrophoresis is commonly used for both analytical and preparative separation of DNA fragments. Standard agarose gels separate DNA fragments from.apprxeq.0.1 to 25 kilobases, or kb, whereas pulsed-field agarose gels resolve molecules from.apprxeq.10 to &gt;2000 kb. Descriptions of standard and pulsed-field agarose gel electrophoresis, as well as parameters affecting resolution of large DNA fragments, are presented in standard textbooks. At least three different protocols employing agarose gels to prepare DNA fragments are known. An agarose gel has the properties of an electric circuit.
One leading protocol for using agarose gel electrophoresis as a simple and highly effective method for the separation, identification and purification of .apprxeq.0. to 25 kb DNA fragments can be divided into three stages. First, a gel is prepared with an agarose concentration appropriate for the size of DNA fragments to be separated. Second, the DNA samples are loaded into the sample wells and the gel is run at a voltage and for a time period that will achieve optimal separation. Third and finally, the gel is stained or, if ethidium bromide has been incorporated into the gel and into the electrophoresis buffer, visualized directly upon illumination with ultraviolet (UV) light.
In order to conduct agarose gel electrophoresis, an agarose gel must be prepared upon a suitable substrate, and made ready to receive the DNA fragments to be separated, normally so as to receive these DNA fragments into each of an arrayed succession of small wells in the form of slits in the agarose gel. This preparation is the subject of the present invention.
2.1 Historical Preparation of Electrophoresis Gels
Preparation typically commences by sealing the edges of a clean, dry, glass plate (or the open ends of the plastic tray supplied with the electrophoresis apparatus) with autoclave tape so as to form a mold. An electrophoresis buffer (usually 1.times.TAE or 0.5.times.TBE) sufficient in amount so as to fill the electrophoresis tank and to prepare a gel is prepared. An amount of powdered agarose is added to a measured quantity of electrophoresis buffer in an Erlenmeyer flask or a glass bottle with a loose-fitting cap. The same batch of electrophoresis buffer is used in both the electrophoresis tank and the gel.
The neck of the Erlenmeyer flask is loosely plugged. The slurry of powdered agarose and buffer is heated in a boilingwater bath or a microwave oven sufficiently so as to permit all of the grains of agarose to dissolve. The dissolved solution is cooled to 60.degree. C., and if desired, ethidium bromide is added.
The liquid is poured into the mold and permitted to cool to produce a gel. The higher the concentration of agarose, the quicker the gel hardens.
In order to produce slits, or wells, in the gel--into which wells samples undergoing electrophoresis will be inserted--a separate comb is typically positioned 0.5-1.0 mm above the glass plate, or base of the mold. The teeth of the comb form a series of linearly aligned wells when the liquid agarose solution is added to the mold. When the comb is properly manually positioned above the plate, or base of the mold, then it is typically supported on its outer teeth so that its inner teeth will automatically lifted slightly above, and clear of, the plate. The present invention will be seen to particularly involve improvements to this comb.
After the gel is completely set both the comb and autoclave tape are carefully removed, and the gel is mounted in the electrophoresis tank. Just enough electrophoresis buffer is added so as to cover the gel to a depth of about 1 mm.
A sample of DNA is mixed with a desired gel-loading buffer. This mixture is slowly loaded into the slits, or wells, of the submerged gel using a disposable micropipette, an automatic micropipettor or a pasteur pipette. Electrophoresis is then performed.
This prior art procedure for the production of a tray of gel, typically an agarose gel, is (i) labor intensive, and thus (ii) expensive, (iii) tedious, and (iv) ill-adapted to efficiencies of scale because a large number of gel trays are roughly more difficult to make than is a single gel tray in proportion to the numbers thereof.
Moreover, the slits, or wells, that are formed in the gel may be of improper or inconsistent depth in accordance that the manual alignment procedure induces errors.
Admittedly, separation by process of electrophoresis of such samples as are later inserted within the slits, or wells, of the gel may accord a certain registration upon viewing (or photographing) that is based on the innate visual properties of the samples. However, it is illogical, and of dubious soundness as a scientific procedure, that successive gels upon which electrophoresis is performed should not be, insofar as is possible, identical. Although exact chemical identity may not be possible between successive gels that are not simultaneously prepared (the present invention will be seen to greatly abet simultaneous preparation of multiple gels), any significant mechanical variation in gel size and thickness, and the locations and depths of the slits (wells) locations in the gels should be, insofar as is possible, strictly avoided.
Presently, the greatest problem occurs with adjusting the depths of the slits should a one, predetermined, slit depth offered by current equipments prove unsatisfactory. Precision machined combs that are matched to gel trays, and which are accurately positionable thereon by action of tongues and grooves, are known. Variable numbers of slits, or wells, may be selected (in accordance that the comb has a greater, or smaller, number of teeth). However, these slits, or wells, are necessarily of differing depths in gels of various thicknesses. Sometimes it is sufficient, and desired, that all slits, or wells, in all of a number of gels should be formed to be at a uniform height, nominally 1 mm, above the base of the tray at their lowermost extension. The present precision gel combs accord this capability. However, it is sometimes desirable to leave a greater thickness below the wells. The present system does not support of creating wells having a variably selectable depth, which depth may, nonetheless to being variably selectable, be uniform from gel tray to gel tray across as large number of gel trays.
2.2 Concurrent Preparation of Multiple Agarose Gels for Electrophoresis in Accordance with the Invention of the Related Application
In accordance with these limitations and problems of the prior art, the related application teaches an apparatus and a method for the simultaneous production of a large number of agarose gel trays complete with precision located and arrayed slits, or wells. These trays are reliably, easily and inexpensively made. The numbers and three-dimensional positions--especially the extent above gel tray bottom--of the slits, or wells, which are formed in the gel are, nonetheless to being variably selectable, precisely controllable. "Precisely controllable" means that two or more gel trays may each be made at separate times to custom mechanical characteristics that are effectively identical. As an arbitrary example, a gel tray having the physical dimensions of 20 cm by 30 cm bed dimension containing a gel of a (relatively thick) 7 mm perforated at exactly 10 slits of 1.0 mm each at positioned 1 cm from a short side wall, each slit to a (relatively high) 1.5 mm above the base of the tray can be repetitively reliably made, and re-made. This precision over a range of dimensions--especially as relate to the vertical extent of the slits above the base of the gel tray--is effectively impossible with the historical apparatus, and procedures.
The related invention and application thus concerns an apparatus and method for greatly (i) reducing the labor, and thus (ii) reducing the expense, (iii) reducing the tedium, and (iv) enhancing the efficiency of producing large numbers of gel trays by virtue of introducing economies of scale to the entire process.
In the related invention, multiple trays of an identical special configuration are vertically stacked, one atop the next. Each tray has and defines a shallow reservoir and an overflow port from the reservoir. The successive stacked trays are in a same top-bottom orientation, but are typically and preferably reversed end-to-end one tray to the next.
A liquid, normally a hot agarose solution, is then poured into the reservoir of the top tray. The liquid fills this top tray's reservoir to a predetermined depth, and then overflows this top tray through its overflow port, flowing downward into a next lower tray in the stack. The liquid continues to overflow each tray in turn, cascading downwards from tray to tray as each tray is filled to a uniform even level and ultimately reaching the bottom tray of the stack. The liquid that is within each tray is permitted to gel, thereby forming a gel state material that is suitable to receive samples upon which electrophoresis may be conducted.
The process may optionally be abetted by the insertion of combination spacer and toothed comb, or spacer-comb, elements between the stacked trays. In its spacer function each element serves to (i) space apart the stacked trays so that any untoward and undesired wicking of liquid onto the underside of an overflowing tray is more effectively precluded. In its simultaneous comb function, each element has and presents teeth that extend downwards into the reservoir of an underlying tray, thereby to (ii) prevent the liquid from occupying an array of small volumes in the reservoir of the tray. By this action, when the liquid gels then an array of small slits, or wells, will have been formed in the gel state material. Samples upon which electrophoresis is performed may be later be inserted into these arrayed wells.
The present invention will shortly be seen to concern still further improvements to these spacer-comb elements.
In particular detail of construction, each tray is preferably substantially in the shape of a nearly flat typically rectangular parallepiped body having and presenting a shallow trough, or reservoir. A tray is typically, and preferably, made from ultraviolet (UV) transparent acrylic plastic. Each tray is suitably sized and configured so as to be vertically stacked with a number of other trays in a vertical stack, and may be so aided by the optional inclusion of interlocking detentes, normally at the tray corners. The trays need not be, and are not normally, stacked in the same orientation in the stack, but are instead reversed 180.degree. in direction along their long axis from one tray to the next.
Each tray has (i) a substantially flat interior reservoir, (ii) an overflow outlet from which liquid received into the reservoir will flow out of the reservoir when a predetermined liquid level is reached in the reservoir, and (iii) a top opening to the reservoir located so that it may be disposed in position below the overflow outlet of any immediately overlying tray in the vertical stack of trays. The trays are commonly rectangular.
The overflow outlet may be in the form of any of (i) a lip, less than the normal height of the sides of the reservoir, that is normally slightly indented from a side of the rectangular tray, (ii) a baffle of predetermined height, less than the walls of the reservoir, that protects a hole that is normally located in a one corner of the reservoir, (iii) a standpipe, typically spaced apart slightly from the sides of the reservoir normally near a corner, that is of lessor height than the walls of the reservoir, or (iv) any mechanical construction that simply permits the reservoir of the tray to fill to a predetermined level and then directs the overflowing fluid downwards from the tray in a preset course.
The top opening of the tray's reservoir may be coextensive with the entire reservoir of the tray itself, or may be more constrained in accordance that the tray's reservoir may be partially covered by an optional cover, and/or an optional spacer-comb element--which elements may have, in some of their configurations, the effect of covering a portion of the tray's reservoir. Regardless of its extent, a top opening to the reservoir is positioned so as to fall in position directly below the egress of overflowing fluid from a superior tray in the vertical stack. The trays so configured permit liquid poured in the topmost tray of a stack to cascade downwards from tray to tray, filling each tray to a predetermined depth, in a similar manner of a classic water clock.
In another of its aspects, the related invention contemplated an apparatus usable so as to prepare from a liquid, normally an agarose gel solution, a flat sheet of a gel state material having and defining an array of wells, wherein each well is suitable to receive a sample upon which electrophoresis may be performed.
The apparatus included (i) a tray having a base and sides defining a substantially flat and shallow interior reservoir; and (ii) a "comb" member at least partially overlying the reservoir of the tray. To the extent the comb member so overlaid the reservoir of the tray it forms a partial cover thereto. The comb member had linearly arrayed teeth in the manner of a comb. The comb member typically and preferably served to interact with mechanical features of the order of guides, or detentes, on the tray in order that it and its teeth may be easily precisely located relative to the tray and the tray's reservoir.
The teeth of the comb member extended, when the comb member was in position overlying the tray and its reservoir, downward into the reservoir. An array of small volumes--corresponding to the volumes occupied by the teeth--was therein established within the reservoir by the teeth of the comb member. No liquid poured or otherwise entered into the tray's reservoir could enter into these volumes because they were occupied by the teeth of the member.
As previously stated, the present invention will shortly be seen to concern still further improvements to these comb members, or spacer-comb elements.
According to the construction of such an apparatus, when a liquid material is poured into the tray while the comb member is in its typically precise location at least partially overlying the tray's reservoir, and after the liquid material is permitted to gel, then a separation of the comb member from the tray left an array of precisely located voids in the gel state material located within the tray's reservoir. These arrayed voids, or slits, or wells, were of suitable size, depth and placement so as to receive samples upon which electrophoresis is performed.
Both aspects of the related invention may beneficially be combined, permitting the quick and efficient preparation of a number of trays, each of which trays contains a gel state material replete with arrayed wells, all at the same time.